The product of the retinoblastoma susceptibility gene, pRB, while functioning as a suppressor of neoplastic cell growth, may also play a wider role as a cell-cycle regulatory element. Cell-cycle analysis of pRB phosphorylation revealed that pRB is wholly unphosphorylated in G1, phosphorylated during S and G2, and becomes dephosphorylated at the onset of M. The current model proposes that pRB function is, at least in part, controlled by its state of phosphorylation. The purpose of the project described here is to take a systematic approach to the identification and localization of pRB modifications and functional domains and to define the relationship between pRB phosphorylation and B-cell lymphoma growth arrest as a consequence of anti-Ig treatment. There are three specific aims designed to accomplish this goal. First, pRB will be chemically cleaved to produce a defined set of ordered polypeptides to localize the phosphorylation domain(s). HPLC analysis will provide a thorough assessment of the amino acids comprising these individual peptides. Peptide-specific antibody generated to the phosphorylation domain(s), metal binding domain, and leucine zipper region, will be used in functional assays designed to block pRB dephosphorylation, divalent cation binding, and pRB complex formation with cellular proteins, respectively. Second, we will isolate and identify the pRB M-phase specific phosphatase and determine how this activity is regulated. Cell extracts will be fractionated and activity monitored by our recently developed in vitro pRB- specific dephosphorylation assay. Regulation of phosphatase activity will be investigated by searching for the presence of regulatory subunits, inhibitory proteins, and enzyme level throughout the cell-cycle. Third, we will define the relationship between the phosphorylation state of pRB and murine B-cell lymphoma growth arrest as a consequence of anti-Ig treatment. We will investigate the possibility that B-cell growth inhibition initiated by IgM signal transduction acts through TGF-beta causing pRB hypophosphorylation and subsequent activation of its growth suppression function. SV40 T-antigen expression, which can override growth arrest by TGF-beta in G1, will be used in experiments to determine if anti- Ig growth arrest can be dissociated from active TGF-beta growth arrest.